Beyond basics: Key mutation selection features for successful tumor-informed ctDNA detection.

Tumor-informed mutation-based approaches are frequently used for detection of circulating tumor DNA (ctDNA). Not all mutations make equally effective ctDNA markers. The objective was to explore if prioritizing mutations using mutational features-such as cancer cell fraction (CCF), multiplicity, and error rate-would improve the success rate of tumor-informed ctDNA analysis. Additionally, we aimed to develop a practical and easily implementable analysis pipeline for identifying and prioritizing candidate mutations from whole-exome sequencing (WES) data. We analyzed WES and ctDNA data from three tumor-informed ctDNA studies, one on bladder cancer (Cohort A) and two on colorectal cancer (Cohorts I and N). The studies included 390 patients. For each patient, a unique set of mutations (median mutations/patient: 6, interquartile 13, range: 1-46, total n = 4023) were used as markers of ctDNA. The tool PureCN was used to assess the CCF and multiplicity of each mutation. High-CCF mutations were detected more frequently than low-CCF mutations (Cohort A: odds ratio [OR] 20.6, 95% confidence interval [CI] 5.72-173, p = 1.73e-12; Cohort I: OR 2.24, 95% CI 1.44-3.52, p = 1.66e-04; and Cohort N: OR 1.78, 95% CI 1.14-2.79, p = 7.86e-03). The detection-likelihood was additionally improved by selecting mutations with multiplicity of two or above (Cohort A: OR 1.55, 95% CI 1. 14-2.11, p = 3.85e-03; Cohort I: OR 1.78, 95% CI 1.23-2.56, p = 1.34e-03; and Cohort N: OR 1.94, 95% CI 1.63-2.31, p = 2.83e-14). Furthermore, selecting the mutations for which the ctDNA detection method had the lowest error rates, additionally improved the detection-likelihood, particularly evident when plasma cell-free DNA tumor fractions were below 0.1% (p = 2.1e-07). Selecting mutational markers with high CCF, high multiplicity, and low error rate significantly improve ctDNA detection likelihood. We provide free access to the analysis pipeline enabling others to perform qualified prioritization of mutations for tumor-informed ctDNA analysis.

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

The Elusive Horizon: Biomarkers in Urothelial Carcinoma.

Urinary tests for circulating tumor DNA have potential for accurate discrimination of bladder cancer from other common inflammatory processes. Efforts are still needed to determine whether these tests can differentiate between cancer and field cancerization and to demonstrate clinical benefit in prospective trials.

Association of Age with Non-muscle-invasive Bladder Cancer: Unearthing a Biological Basis for Epidemiological Disparities?

BACKGROUND: Age disparity in patients with non-muscle-invasive bladder cancer (NMIBC) exists. Whether this is due to differences in adequate cancer care or tumour biology is unclear. OBJECTIVE: To investigate age disparities in NMIBC using the Surveillance, Epidemiology, and End Results (SEER)-Medicare and UROMOL datasets. DESIGN, SETTING, AND PARTICIPANTS: The SEER-Medicare data were used to identify patients with clinical stage Ta, Tis, and T1 NMIBC between 2005 and 2017 (n = 32 225). Using the UROMOL cohort (n = 834), age disparities across transcriptomic, genomic, and spatial proteomic domains were assessed. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: For the SEER-Medicare data, multivariable competing-risk regression was used to examine the association between age and recurrence, progression, and bladder cancer-specific mortality (BCSM). For the UROMOL cohort, multivariable general linear model and multinomial logistic regression were performed to evaluate the association between age and tumour biology. RESULTS AND LIMITATIONS: An analysis of the SEER-Medicare cohort revealed 5-yr recurrence rates of 55.2%, 57.4%, and 58.9%; 5-yr progression rates of 25.6%, 29.2%, and 36.9%; and 5-yr BCSM rates of 3.9%, 5.8%, and 11.8% in patients aged 66-70, 71-80, and ≥81 yr, respectively. After multivariable adjustment, age ≥81 yr was associated with a higher risk of recurrence (hazard ratio [HR] 1.07, 95% confidence interval [CI] 1.03-1.12; p = 0.001), progression (HR 1.32, p < 0.001), and BCSM (HR 2.58, p < 0.001). UROMOL2021 transcriptomic class 2a was most frequently observed in patients with advanced age (34.0% in ≥76 yr vs 21.6% in ≤65 yr; p = 0.004), a finding confirmed on multivariable analysis (risk ratio [RR] 3.86, p = 0.002). UROMOL2021 genomic class 3 was observed more frequently in patients aged ≥76 yr (4.9% vs 24.2%; p = 0.001). Limitations include the definitions used for recurrence and progression, which may lead to under- or overestimation of true rates. CONCLUSIONS: Among SEER-Medicare patients with NMIBC, advanced age is associated with inferior oncological outcomes. These results reflect age-related molecular biological differences observed across transcriptomic and genomic domains, providing further evidence that innate tumour biology contributes to observed disparities in NMIBC outcomes. PATIENT SUMMARY: Older patients with non-muscle-invasive bladder cancer have worse oncological outcomes than younger patients. Some of this age disparity may be due to differences in tumour biology.

Multiomics profiling of urothelial carcinoma in situ reveals CIS-specific gene signature and immune characteristics.

Urothelial carcinoma in situ (CIS) is an aggressive phenotype of non-muscle-invasive bladder cancer. Molecular features unique to CIS compared to high-grade papillary tumors are underexplored. RNA sequencing of CIS, papillary tumors, and normal urothelium showed lower immune marker expression in CIS compared to papillary tumors. We identified a 46-gene expression signature in CIS samples including selectively upregulated known druggable targets MTOR, TYK2, AXIN1, CPT1B, GAK, and PIEZO1 and selectively downregulated BRD2 and NDUFB2. High expression of selected genes was significantly associated with CIS in an independent dataset. Mutation analysis of matched CIS and papillary tumors revealed shared mutations between samples across time points and mutational heterogeneity. CCDC138 was the most frequently mutated gene in CIS. The immunological landscape showed higher levels of PD-1-positive cells in CIS lesions compared to papillary tumors. We identified CIS lesions to have distinct characteristics compared to papillary tumors potentially contributing to the aggressive phenotype.

Optimizing the Use of Next-Generation Sequencing Assays in Patients With Urothelial Carcinoma: Recommendations by the 2023 San Raffaele Retreat Panel.

BACKGROUND: The application of precision medicine in clinical practice implies a thorough evaluation of actionable genomic alterations to streamline therapeutic decision making. Comprehensive genomic profiling of tumor via next-generation sequencing (NGS) represents a great opportunity but also several challenges. During the 2023 San Raffaele Retreat, we aimed to provide expert recommendations for the optimal use of NGS in urothelial carcinoma (UC). MATERIALS AND METHODS: A modified Delphi method was utilized, involving a panel of 12 experts in UC from European and United States centers, including oncologists, urologists, pathologists, and translational scientists. An initial survey, conducted before the meeting, delivered 15 statements to the panel. A consensus was defined when ≥70% agreement was reached for each statement. Statements not meeting the consensus threshold were discussed during the meeting. RESULTS: Nine of the 15 statements covering patient selection, cancer characteristics, and type of NGS assay, achieved a consensus during the survey. The remaining six statements addressing the optimal timing of NGS use, the ideal source of tumor biospecimen for NGS testing, and the subsequent need to evaluate the germline nature of certain genomic findings were discussed during the meeting, leading to unanimous agreement at the end of the conference. CONCLUSION: This consensus-building effort addressed multiple unanswered questions regarding the use of NGS in UC. The opinion of experts was in favor of broader use of NGS. In a setting where recommendations/guidelines may be limited, these insights may aid clinicians to provide informed counselling and raise the bar of precision and personalized therapy.

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

Whole-genome Mutational Analysis for Tumor-informed Detection of Circulating Tumor DNA in Patients with Urothelial Carcinoma.

BACKGROUND AND OBJECTIVE: Circulating tumor DNA (ctDNA) can be used for sensitive detection of minimal residual disease (MRD). However, the probability of detecting ctDNA in settings of low tumor burden is limited by the number of mutations analyzed and the plasma volume available. We used a whole-genome sequencing (WGS) approach for ctDNA detection in patients with urothelial carcinoma. METHODS: We used a tumor-informed WGS approach for ctDNA-based detection of MRD and evaluation of treatment responses. We analyzed 916 longitudinally collected plasma samples from 112 patients with localized muscle-invasive bladder cancer who received neoadjuvant chemotherapy (NAC) before radical cystectomy. Recurrence-free survival (primary endpoint), overall survival, and ctDNA dynamics during NAC were assessed. KEY FINDINGS AND LIMITATIONS: We found that WGS-based ctDNA detection is prognostic for patient outcomes with a median lead time of 131 d over radiographic imaging. WGS-based ctDNA assessment after radical cystectomy identified recurrence with sensitivity of 91% and specificity of 92%. In addition, genomic characterization of post-treatment plasma samples with a high ctDNA level revealed acquisition of platinum therapy-associated mutational signatures and copy number variations not present in the primary tumors. The sequencing depth is a limitation for studying tumor evolution. CONCLUSIONS AND CLINICAL IMPLICATIONS: Our results support the use of WGS for ultrasensitive ctDNA detection and highlight the possibility of plasma-based tracking of tumor evolution. WGS-based ctDNA detection represents a promising option for clinical use owing to the low volume of plasma needed and the ease of performing WGS, eliminating the need for personalized assay design. PATIENT SUMMARY: Detection of tumor DNA in blood samples from patients with cancer of the urinary tract is associated with poorer outcomes. Disease recurrence after surgery can be identified by the presence of tumor DNA in blood before it can be detected on radiography scans.

Immune Contexture Changes Following Blue Light Cystoscopy with Hexaminolevulinate in Bladder Cancer.

Background: Transurethral resection of bladder tumor (TURBT) is a central component in the diagnosis of non-muscle-invasive bladder cancer (NMIBC) and can be guided by several optical imaging techniques for better visualization of lesions. Objective: To investigate if a change in tumor microenvironment (TME) composition could be observed as an effect of hexaminolevulinate (HAL)-assisted blue light cystoscopy (BLC) in TURBT samples from patients with bladder cancer. Design setting and participants: This was a retrospective study of 40 patients with bladder cancer who underwent either BLC-guided TURBT (n = 20) or white light cystoscopy (WLC)-guided TURBT (n = 20) before radical cystectomy (RC). Tissue samples (n = 80) were collected from paired TURBT and RC specimens for all 40 patients. Tumor tissue was stained using multiplex immunofluorescence and immunohistochemistry. Outcome measurements and statistical analysis: Immune cell infiltration was assessed according to the proportions of each immune cell or immune evasion marker and the relative change from TURBT as baseline was calculated. Statistical comparisons between groups were performed using the Wilcoxon rank-sum test or the paired-sample Wilcoxon test. Results and limitations: Comparison of relative changes in the TME revealed a significant decrease in stromal infiltration of cytotoxic T cells (p = 0.024), B cells (p = 0.041), and stromal cells expressing PD-1 (p = 0.011) in patients treated with BLC-guided TURBT compared to WLC-guided TURBT. Conclusions: Our pilot study showed that HAL-BLC during TURBT in bladder cancer may influence the immune cell composition and TME. Patient summary: We investigated the potential therapeutic effect of blue light versus white light for guidance in removing bladder tumors via the urethra in patients with bladder cancer. For blue light guidance, a compound called hexaminolevulinate is used to visualize tumor tissue. We found changes in immune cell composition that may have been influenced by the blue light guidance.

An Association Study of Germline Variants in Bladder Cancer-Related Genes with the Prognosis of Non-Muscle Invasive Bladder Cancer.

BACKGROUND: Various germline genetic variants are associated with the prognosis of non-muscle invasive bladder cancer (NMIBC). Germline variants in genes frequently somatically mutated in bladder cancer have not been studied thoroughly in relation to risk of recurrence or progression in NMIBC. OBJECTIVE: To identify germline DNA variants in bladder carcinogenesis-related genes associated with recurrence or progression in NMIBC. METHODS: We analysed associations between single-nucleotide polymorphisms (SNPs) and NMIBC recurrence and progression using data from the Nijmegen Bladder Cancer Study (NBCS, 1,443 patients). We included 5,053 SNPs within 46 genes known to have mutation, overexpression or amplification in bladder cancer. We included all recurrences in the statistical analysis and performed both single variant analysis and gene-based analysis. SNPs and genes that showed significant or suggestive association (false discovery rate P value < 20%) were followed-up in independent cohorts for replication analysis, through eQTL analysis and tests for association of tumour expression levels with NMIBC recurrence and progression. RESULTS: Single variant analysis showed no statistically significant associations with recurrence or progression. In gene-based analysis, the aggregate effect of the 25 SNPs in the Cyclin D1 gene (CCND1) was statistically significantly associated with NMIBC recurrence (Punadj = 0.001, PFDR = 0.046), but not with progression (Punadj = 0.17, PFDR = 0.54). Validation analysis in independent cohorts did not confirm the association of CCND1 with NMIBC recurrence. CONCLUSIONS: We could not identify reproducible associations between common germline variants in bladder carcinogenesis-related genes and NMIBC recurrence or progression.

Sensitivity and Specificity in Urine Bladder Cancer Markers - Is it that Simple?

Marker research, and in particular urine bladder cancer marker research throughout the past three decades, devours enormous scientific resources in terms of manpower (not to mention time spent on reviewing and editorial efforts) and financial resources, finally generating large numbers of manuscripts without affecting clinical decision making. This is mirrored by the fact that current guidelines do not recommend marker use due to missing level 1 evidence. Although we recognize the problems and obstacles, the authors of this commentary feel that the time has come to abandon the current procedures and move on to prospective trial designs implementing marker results into clinical decision making. Our thoughts and concerns are summarized in this comment.